Recombinant mycobacterial DNA-binding protein 1 with post-translational modifications boosts IFN-gamma production from BCG-vaccinated individuals’ blood cells in combination with CpG-DNA

Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis.

www.nature.com/scientificreports/We immunized BALB/c mice with native MDP1 purified from BCG and screened for Ig-producing B cell hybridoma in lymph nodes and immortal myeloma cells.One isolated hybridoma (7C4C3D4) produced anti-MDP1 IgG, which was designated 7C.We identified that the epitope recognized by mAb7C is located in the 41-60 amino acid region of MDP1, because synthetic peptides corresponding to this region inhibited MDP1-mAb 7C interaction (supplementary Fig. S1A and B).The 41-60 amino acid region of BCG-MDP1 is consistent with that of M. tuberculosis-MDP1 (Supplementary Fig. S1 C).
To screen for hybridomas producing antibodies that recognize dimethylated lysin, we synthesized peptides with the same amino acid sequence as immunized peptide, with dimethylation of lysin at the 10th position, at the 11th position, and at both the 10th and 11th positions, and conducted ELISA (Supplementary Fig. S2A and  B).We obtained one hybridoma, designated 38-8, produces IgG only recognizes the dimethylation of 10th , 11th or both lysine residues (Supplementary Fig. S2B).
We conducted Surface Plasmon Resonance (SPR) analysis to determine the binding kinetics of mAb 7C and 38-8 mAb to MDP1.The dissociation constant (KD) values were 3.08 × 10 -10 M and 7.56 × 10 -9 M, respectively (Supplementary Fig. S1D and S2C).These mAbs were utilized for detection of MDP1 in our research.

Preparation of MDP1 produced in E. coli (eMDP1)
Recombinant MDP1 produced in E. coli as histidine-tag proteins, which was designated eMDP1, can be purified by previously established methods 13 .However, its purification efficiency was 0.1 mg from 1 L culture of recombinant E. coli.
One possible reason for the low yield might be low expression levels due to different codon usage between E. coli and M. tuberculosis.Another possible reason is unstable mRNA level due to degradation of foreign genederived mRNA.As such, we next tested these hypotheses in order to solve the problem of low eMDP1 yield.First, we used a synthetic gene whose codons were optimized to that of E. coli (Supplementary Fig. S3).Second, we used Rosetta (DE3) E. coli that genetically complemented transfer RNAs of rare codons.In the third option, we used BL21Star (DE3) which stabilizes mRNA.Each recombinant E. coli containing MDP1-expression cassette was cultured in LB media and eMDP1 expression was induced by IPTG 13 .
We analyzed eMDP1-expression levels by SDS-PAGE and western blot using mAb 7C or anti-6-HIS antibody.We found elevated eMDP1-expression only when we used Rosetta (DE3) E. coli (Supplementary Fig. S4).This suggests that rare codon in M. tuberculosis-MDP1 gene can be the bottleneck for MDP1 expression in E. coli.
Based on these data, we decided to use Rosetta (DE3) E. coli as the host cell for eMDP1 purification.As a result, we could purify 1.0 mg of eMDP1 from 1L culture of Rosetta (DE3) E. coli.
To express M. tuberculosis-MDP1 in pSO246-ACE-hupB-His/M.smegmatis, we cultured the transformant in Mueller Hinton II media until optical density at 600 nm (OD 600 ) of 1.0 and then added acetamide (ACE), an activator of the acetamidase promoter.Forty-eight hours later, we saw a substantially increased protein band at 28 kDa (Fig. 1B).In the western blot analysis, the same band was recognized by both mAb 7C and mAb against 6X His-Tag (Fig. 1B), showing substantial expression of M. tuberculosis-MDP1 by addition of ACE in recombinant M. smegmatis.
Next, we searched for optimal time points feasible for mMDP1 purification.mMDP1 was identified in the insoluble fraction of the bacterial lysate following lysis with the BugBuster bacterial lysis reagent (Fig. 1C).The amount of mMDP1 was comparable in lysates obtained at 24, 48, and 72 h after addition of ACE (Fig. 1D, left).We found that mAb 38-8, which recognizes post-translational modifications, reacted with mMDP1.Its reaction level was highest in 72 h after addition of ACE (Fig. 1D, right).However, proteins started degradation probably due to entering the late-stationary phase.We thus chose 48 h incubation with ACE for mMDP1 purification.
We next proceeded with the purification of mMDP1.mMDP1 was not solubilized with commercially available bacterial lysis buffer, the BugBuster reagent (Fig. 1C).Therefore, we employed the acid-extraction method with 0.25 M HCl, which was used to solubilize mycobacterial MDP1 11 .After neutralization, acid solubilized proteins including mMDP1 were denatured by dialyzing in 6 M Urea and applied to a nickel column under the control of AKTA explore FPLC system (Supplementary Fig. S5A).Fractions containing MDP1 were refolded on the nickel column by gradual reducing the Urea concentration from 6 to 0 M (Methods and Supplementary Fig. S5B).We obtained 2.3 mg of mMDP1 from 1 L culture of pSO246-ACE-hupB-His/M.smegmatis.Thus, the purification efficacy was higher when using M. smegmatis as a host cell compared to E. coli (1 mg/L).www.nature.com/scientificreports/ We next examined the oligomerization status of eMDP1 and mMDP1.Sedimentation velocity (SV) analysis revealed that both eMDP1 and mMDP1 exist as monomers at physiological NaCl concentrations (150 mM), and in 500 mM NaCl (Fig. 2B).
We also performed glutaraldehyde crosslinking analysis to judge the association time between molecules 27 .As expected from the SV measurement data, most eMDP1 and mMDP1 were monomers.Vast majority of eMDP1 were not crosslinked and fractionated as monomers after glutaraldehyde crosslinking, although dimers were detected at 4 µM eMDP1 at 1 M salt concentration and at 8 µM eMDP1 at all salt concentrations (Fig. 2 C left panels).In contrast, part of mMDP1 was crosslinked at all tested salt concentration, and dimeric and trimeric crosslinked forms increased at higher salt concentrations (Fig. 2C right panels).The glutaraldehyde crosslinking study suggests that the association time between proteins was longer for mMDP1 than for eMDP1.
Taken together these results indicate that one, most eMDP1 and mMDP1 are monomers at physical condition, two, the dimer/monomer ratio increase in the salt concentration for both eMDP1 and mMDP1, and three, mMDP1 tends to oligomerize more frequently than eMDP1.

mMDP1 stimulates IFN-gamma production by peripheral blood cells derived from BCG-vaccinated individuals
Th1-type cells produce IFN-gamma which is critical for vaccine-induced protection against tuberculosis both in mice and human 25,26 .To assess the feasibility of eMDP1 and mMDP1 as booster vaccine antigens, we next evaluated their ability to stimulate IFN-gamma production by peripheral blood mononuclear cells (PBMC) or whole blood cells derived from BCG-vaccinated individuals.The selected donors received BCG (BCG Tokyo 172) vaccine over 20 years ago, and it is believed that the efficacy of BCG has since diminished 28 .
PBMC from 3 individuals was cultured with either eMDP1 or mMDP1 followed by quantification of IFNgamma levels in culture supernatants.We found that all tested PBMC from 3 individuals produced higher amounts of IFN-gamma when stimulated by mMDP1 than by eMDP1 (Fig. 3).These data show stronger induction of IFN-gamma production by mMDP1 than eMDP1 in BCG-vaccinated individuals.

Comparison of immuno-stimulatory efficacy of MDP1 to other major M. tuberculosis antigens
Several immunodominant proteins are proposed vaccine candidates against tuberculosis 29 .More attention is focused on secreted proteins as they are likely to efficiently stimulate immune cells compared to non-secreted proteins 30 .We therefore evaluated the immunogenicity of mMDP1 to comparable to that of secreted M. tuberculosis proteins.
We incubated each purified secreted protein or mMDP1 with bloods derived from 5 healthy individuals who were vaccinated with BCG more than 20 years ago, and measured IFN-gamma production.We found heterologous IFN-gamma production among donors (Fig. 4A).However, the level of IFN-gamma production in blood stimulated with mMDP1 was higher than when stimulated with any secreted proteins including immunodominant proteins such as MPT32, antigen 85B (MPT59) and C (MPT45), except in the case of MPT32 for 1 donor (Fig. 4A).These data suggest that mMDP1 is more immunogenic than secreted M. tuberculosis proteins in BCG-vaccinated individuals (Fig. 4B).

Determination of mMDP1 post-translational modification
Our data shows that mMDP1 is more immunogenic than eMDP1.This could be due to post-translational modifications in mMDP1, which are absent in eMDP1.Although mAb 38-8 recognizes mMDP1 but not eMDP1 (Fig. 1D, right), the entire post-translational modifications on mMDP1 are unknown.We next identified posttranslational modifications in mMDP1 by utilizing a newly developed method using limited proteolysis with trypsin in combination of liquid chromatography-electrospray tandem mass spectrometry (LC/MS) 33 .The data Expression cassette of M. tuberculosis MDP1 gene (hupB) with His-6 tag driven by an M. smegmatis acetamidase promoter was inserted into E. coli-mycobacteria shuttle vector pSO246.(B) Induction of M. tuberculosis-MDP1 expression in pSO246-ACE-hupB-His/M.smegmatis.pSO246-ACE-hupB-His/M.smegmatis was cultured for 48 h in the presence or absence of 2% ACE.Ten µg of lysate was fractionated by SDS-PAGE in 12% acrylamide gel and visualized by CBB staining (left).For western blot, 2 µg of bacterial lysate was fractionated and transferred to a membrane, followed by incubation with anti-MDP1 mAb 7C (middle) or anti-His-Tag antibody (right).M: molecular marker, (−): absence of acetamide, ( +): presence of acetamide.(C) Time course and soluble fraction of mMDP1.Harvested pSO246-ACE-hupB-His/M.smegmatis at 0, 24, 48, and 72 h after addition of ACE were treated with bacteria-disrupting agent, BugBuster.Five µg of each soluble (sup) and insoluble (ppt) fraction was fractionated by 12% SDS-PAGE and stained with CBB. a and b, 300 ng of native MDP1 purified from M. tuberculosis H37Rv and eMDP1, respectively.(D) Detection of MDP1 expression by mAb 7C and mAb 38-8.mMDP1 was purified from pSO246-ACE-hupB-His/M.smegmatis at 0, 24, 48, and 72 h after addition of ACE.Western blot was then carried out by using mAb 7C (left) and mAb 38-8 (right).One hundred ng of protein and 50 ng of eMDP1 was loaded in each lane.(E) Chemical methylation of eMDP1.eMDP1 was treated with methylation reagent for 0, 30, 90 min.Western blot analysis was conducted using an anti-His antibody (left) and mAb38-8 (right).One hundred ng of protein was loaded per lane.cont: mMDP1.www.nature.com/scientificreports/shows that, compared to eMDP1, most of the lysine residues on the C-terminal half of mMDP1 were highly methylated, moderately dimethylated, trimethylated, and acetylated 34 (Fig. 5).Importantly, the pattern of posttranslational modifications in mMDP1 closely resemble that of wild-type M. tuberculosis MDP1 33 , which might explain high antigenicity of mMDP1 in BCG-vaccinated individuals.

Evaluation of adequate CpG-ODN adjuvant for MDP1
Synthetic oligonucleotides containing CpG motifs (CpG ODN) are considered ideal adjuvants for MDP1, since they increase immunogenicity of MDP1 11 .We next sort to determine the suitable CpG ODN to increase the antigenicity of MDP1.CpG-DNAs are categorized into 3 classes based on their structure.We used ODN 2216 (Class A), ODN 2006 (Class B), and ODN 2395 (Class C) in this study.In addition to these representative CpG-DNAs, we used a new class of CpG-ODN designated as G9.1, that we recently developed 23 (Supplementary Table 1).As control, we used their negative sequence forms-CpG motif was changed to GpC-as control (Supplementary Table 1).
We measured IFN-gamma production from whole blood sample obtained from a healthy BCG-vaccinated individual after stimulation with mMDP1 or a combination of mMDP1 and each ODN as adjuvant (Fig. 6).ODNs at concentrations ranging from 0.01 M to 1 µM induced little IFN-gamma production (Fig. 6A).When blood was cultured with 0.5 µM of mMDP1 alone, 302 ± 29.9 pg/ml of IFN-gamma production was observed (Fig. 6C).Combination of mMDP1 with either Class B, Negative form (Neg) Class B, Class C, or Neg Class C did not exceed stimulatory effects by mMDP1 alone.In contrast, the combination of mMDP1 with G9.1 or ODN 2216 (Class A) produced more IFN-gamma than mMDP1 alone.The highest IFN-gamma production was observed by the combination of mMDP1 and G9.1 (1180 ± 258 pg/ml) (Fig. 6B).
We increased the number of BCG-vaccinated donors to confirm augmentation of MDP1-induced IFNgamma production by G9.1 (Fig. 7A).The combination of mMDP1 and G9.1 induced significantly higher IFNgamma production than mMDP1 alone, and the combination of eMDP1 and G9.1.Unexpectedly, combination of mMDP1 and Neg G9.1, non CpG-DNA also induced more IFN-gamma production than mMDP1 alone (Fig. 7A).
It is reported that, in human peripheral blood cell populations, B cells, monocytes, and plasmacytoid dendritic cells (pDC) expresses TLR9, the non-methylated CpG-DNA receptor 20,35,36 .Among these cells, pDC produces massive amount of IFN-alpha in response to CpG-ODNs 37 .As such, we next tested IFN-alpha production from blood cells in the same experimental setting.We observed remarkable production of IFN-alpha when blood samples were stimulated with a combination of eMDP1 or mMDP1 and G9.1.However, mMDP1 exhibited a tendency to be more immunogenic.In contrast, IFN-alpha production when eMDP1 or mMDP1 was combined with Neg G9.1 resulted in lower IFN-alpha production compared to the combination with G9.1.This suggests that TLR9 stimulation is required for IFN-alpha production (Fig. 7B).These results show the importance of TLR-9 for IFN-alpha production by pDC stimulated with MDP1-G9.1 complex.

mMDP1 binds more strongly to G9.1 than Class A, Class B, and Class C ODNs
We observed synergistic effects on immune-stimulation by combination of mMDP1 with G9.1, Neg G9.1, or ODN 2216 but not with other tested CpG-ODNs.Since MDP1 is a DNA-binding protein, we examined whether mMDP1 binds directly to each CpG-ODN using Bio-Layer Interferometry.
We found that mMDP1 bound to G9.1 and Neg.G9.1 more strongly than ODN 2216 (Class A) and ODN 2006 (Class B). mMDP1 did not bind to ODN 2395 (Class C).The affinity of mMDP1 with G9.1 and Neg G9.1 were similar and calculated to be 6.73 × 10 -9 M, 4.93 × 10 -9 M. On the other hand, the affinity of mMDP1 with Class A (2.50 × 10 -8 M) and Class B (5.83 × 10 -8 M) were lower (Supplementary table 2).These results suggest that the heightened levels of IFN-gamma production by immune cells stimulated by the combination of mMDP1 and CpG-DNAs are correlated with their mutual affinity.

G9.1 promotes binding of MDP1 to PBMC
Binding to cell surface receptors is the initial step for immune activations by ligands.In order to identify the mechanism of synergistic immune-stimulation by mMDP1 and G9.1, we evaluated interaction of mMDP1, G9.1, and mMDP1-G9.1 mixture with PBMC by flow cytometry analysis.www.nature.com/scientificreports/ We found that majority of MDP1, G9.1, and mMDP1-G9.1 mixture bound to the cells in the lineage plus and HLA-DR plus region, which mediates antigen presentation to T cells in the periphery rather than dendritic cells (Fig. 8B, middle and right column).The percentage of cells bound to mMDP1 increased with the addition of G9.1 compared to mMDP1 alone (Fig. 8C).These results indicate that mMDP1-G9.1 complex can more efficiently access antigen presenting cells compared to its individual components.This finding partially explains their synergistic immune stimulations of human PBMC.

Discussion
BCG, the tuberculosis vaccine, is administered from the neonatal period to early childhood.While it is effective against tuberculosis in childhood, its effectiveness gradually wanes with age.This study aimed to develop a booster tuberculosis vaccine for individuals who have already been BCG-vaccinated.MDP1 is a major M. tuberculosis protein and a candidate for booster vaccine component 22,38 .Here we evaluated its production, antigenicity, immunogenicity, and optimized its accompanying adjuvant.
We first discovered that using Rosette (DE3) E. coli supplemented with tRNAs for AGG, AGA, AUA, CUA, CCC, and GGA codons increased MDP1 expression.This suggest that for E. coli, MDP1 contains rare codons which can partly explain its low expression in E. coli (Figs S3, and S4).We next observed that larger amounts of MDP1 could be purified from M. smegmatis compared to Rosette (DE3) E. coli.This suggests that, for expression of MDP1, the intracellular environment in M. smegmatis is more suitable than that in E. coli.These findings highlight the merit of using M. smegmatis not only for the expression of MDP1 but also for other mycobacterial proteins.
Monoclonal antibody 38-8 (mAb 38-8), which reacts to the 146th and 147th dimethylated lysins of MDP1, recognizes mMDP1 expressed in M. smegmatis, indicating that these two lysines are dimethylated (Fig. 1D and S Fig. 2B).M. tuberculosis MDP1 contains as many as 41 residues of lysin, but the details of their posttranslational modification have not been revealed due to its complexity, which resists conventional analysis, although only acetylation of MDP1 was reported 14 .We recently established a method to determine multiple post-translational modifications on a protein 33 .By employing this method, we revealed that both native MDP1 (nMDP1) and mMDP1 exhibit highly similar lysin methylation patterns at the C-terminal regions 33 .Although the methyltransferase responsible for MDP1 methylation has not yet been identified, these results suggest that the methyltransferases involved in the methylation of lysine residues on MDP1 in both M. tuberculosis and M. smegmatis share the same or very similar substrate specificity 17 .This implies that M. smegmatis can produce MDP1 with the same physical characteristics as that produced by M. tuberculosis.
We acknowledge some limitations in our study.Firstly, The number of participants is small because of single center study.Second, diagnosis of HIV infection nor QFT test to clarify latent tuberculosis infection (LTBI) was not conducted.Thirdly, the infection with NTM was not confirmed through antibody testing.However, we found that post-translationally modified mMDP1 has much stronger immunogenicity than eMDP1.mMDP1 elicits a larger production of IFN-gamma by PBMCs derived from BCG-vaccinated individuals (Figs. 3 and 7).The immunogenicity of mMDP1 is comparable to or greater than that of the tested secreted proteins (Fig. 4).This may be because some MDP1 is also present on the cell wall, where it is easily recognized by immune cells 39 .This strong mMDP1 immunogenicity in BCG-vaccinated individuals indicates its potential use as a booster vaccine antigen.
MDP1 post-translational modifications produced by BCG have not been analyzed, but in view of the concordance between the modification patterns of mMDP1 and nMDP1, it is likely that similar modifications are occurred on MDP1 produced by BCG.Cells that produced IFN-gamma in response to mMDP1 stimulation (Figs. 3 and 7) are thought to be T cells that were activated after donors were vaccinated with BCG.Therefore, the heightened immunogenicity of mMDP1 compared to eMDP1 in donors is likely attributed to IFN-gammaproducing T cells recognizing the post-translationally modified MDP1 produced by BCG.Additionally, we successfully established mAb 38-8, which specifically recognizes post-translationally modified MDP1.Thus, this study confirms that both T and B cells can accurately recognize the post-translationally modified polypeptides.
Post-translational modifications alter or confer functions of proteins and, in some cases, stabilize the protein structure.They also mark proteins for degradations.In this way, there are many proteins that are posttranslationally modified in living organisms.On the other hand, immune responses specifically recognize the antigens of pathogens with accuracy.Therefore, post-translationally modified polypeptides are also targets for recognition by immune responses.Besides HBHA and MDP1, there have been reported to exhibit post-translationally modified proteins in M. tuberculosis.Thus, ESAT6 is acetylated, Apa/Rv1860/MPT32 and PstS1 are O-glycosilated protein, and LpqH is lipoproteins 40 .Interestingly all of these post-translationally modified proteins show strong immunogenicity.ESAT6 is a major virulence factor of M. tuberculosis, which is lacking in an attenuated strain, BCG.Acetylation of Ser and Thr resides in ESAT6 has been documented 41 .Interestingly, native ESAT6 from M. tuberculosis showed efficacy comparable to BCG against M. tuberculosis infection, whereas rESAT6 produced in E. coli showed no protective effect 42 .Apa is a mannosylated protein 40 .Stimulation with mannosylated native Apa has been shown to enhance the production of cytokines such as IL-4, IL-17, and IFN-gamma from BCG vaccinated human PBMCs and spleen or lung cells of BCG-infected mice 43 .This mannosylated Apa-peptide was indeed recognized by an established HLA-E-specific human CD8 + T cell 44 .LpqH lipoprotein is recognized through toll-like receptor 2 and triggers Th1 responses 45 .PstS1 was observed the immunodominant 38 kDa protein 46 and a recent report suggested anti-PstS1 antibodies contribute to host protection in humans 47 .We recently reported antibody levels against native M. tuberculosis proteins including Apa and PstS1, but the corresponding E. coli recombinant proteins were unable to detect pulmonary tuberculosis patients 32 .Exact reasons for the strong immunogenicity of post-translationally modified proteins of M. tuberculosis can be complex, but this is fundamentally due to accuracy of immune responses.Thus, taking into consideration of the native structure of www.nature.com/scientificreports/antigens is important for understanding immune responses and developing relevant attractive strategies, such as, vaccines and immunotherapies.MDP1 and CpG-DNA synergistically enhance each other's immunogenicity 22 .Therefore, to utilize mMDP1 as a vaccine component, we examined the adjuvant effects of type A-C and G9.1 CpG-DNA.The results showed that G9.1 strongly enhanced the immunogenicity of mMDP1.This enhancement was attributed to the strength of their binding, as observed in the analysis using Bio-Layer Interference (Sup Table 2).These results suggest that the immunogenic enhancement of MDP1 by CpG-DNA depends on their affinity.This is reasonable, as MDP1 can be efficiently presented by antigen-presenting cells activated by the CpG-DNA simultaneously.Indeed, the primary cells taking up mMDP1 were the HLA-DR positive cell population, which possesses antigen-presenting capacity, and the uptake rate was further increased by the addition of G9.1 (Fig. 8).This may explain the synergistic immune activation of MDP1 and G9.1.
The results of this study demonstrate important implications for the development of component vaccines.Specifically, structural information of antigens produced by pathogens should be considered in vaccine design.This is because proteins from live vaccines and pathogens may have unique post-translational modifications, which are well recognized by the host's immune system 16 .In the development of viral vector or mRNA vaccines using M. tuberculosis antigens, the specific post-translational modifications of M. tuberculosis are not taken into account.In such cases, the structure of the antigen produced by the vaccine may differ, even if the amino acid sequence is identical.
In this study, we investigated the potential of MDP1 as a booster vaccine, primarily focusing on its ability to enhance IFN-gamma production.We specifically explored the immunogenicity of MDP1 produced by M. smegmatis(mMDP1), in combination with G9.1-CpG-DNA.This combination showed promise in stimulating IFN-gamma production from immune cells in BCG-vaccinated individuals, although we didn't quantify the number of IFN-gamma producing cells using flow cytometry or ELISPOT assays.IFN-gamma is a crucial cytokine in the defense against tuberculosis (TB), and its production is considered vital for an effective TB vaccine [24][25][26] .However, it's important to note that excessive production of IFN-gamma can lead to tissue destruction 48,49 .Moreover, relying solely on IFN-gamma production as a marker of vaccine efficacy may not be sufficient.Recent reports have highlighted the importance of considering non-Th1 cytokines 50 , antibody levels 51 , and innate immune activation at the time of vaccination 52 as additional indicators of vaccine efficacy.While the mMDP1 and G9.1 combination appears promising as a booster vaccine for TB, it is imperative to investigate other markers beyond IFN-gamma productivity.In addition, we've not evaluated real efficacy of booster vaccination of mMDP1 and G9.1 in animal models and BCG-primmed individuals.We previously reported that even eMDP1 plus G9.1 booster immunization was effective against M. tuberculosis challenge in BCG-primed guinea pig 22 .It is reasonable to next investigate whether mMDP1 is superior to eMDP1 in BCG-primed guinea pig model before starting clinical study.Three days after the additional immunization (17 days after the initial immunization), cells were harvested from ilioinguinal lymph nodes and then, fused with myeloma SP2 cells.To examine m38-8 recognition sites, ELISA test was performed by using four different peptides (dimethylated 10th lysine, dimethylated 11th lysine, both 10 and 11 dimethylated, and unmodified) conjugated with BSA.These were plated on flat-bottom plate (3 µg/ ml, 50 µl/well), and ELISA tests were performed with the addition of hybridoma culture supernatant.Affinity of dimethylated mMDP1 and m38-8 was measured by Biacore.The measurement method was the same as 7C.

Determination of Mtb MDP1 (mMDP1) protein expression levels
An aliquot of cell pellet was first washed with distilled water.Glass beads (1 mm diameter, Kishida Chemical, Osaka, Japan) and Bugbuster HT Protein Extraction Reagent (Merck KGaA, Darmstadt, Germany) were then added to the cell pellet which was then disrupted with BeadSmash BS-12R (Wakenyaku, Kyoto, Japan) at 5500 rpm, 4 °C for 30 s three times with 30 s intervals between operation.Samples were then centrifuged at 14,000 rpm for 5 min at 4 °C.This was followed by separate collection of the supernatant and insoluble fractions, which were then treated with Laemmli sample buffer at 95 °C for 10 min.In some experiment, cells were disrupted with glass beads and water, and then treated with Laemmli sample buffer at 95 °C for 10 min.After centrifuging at 14,000 rpm for 5 min, the supernatant (whole cell lysate) was obtained.Sample's protein concentrations were measured by Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Waltham MA).The samples were electrophoresed on a sodium dodecyl sulphate (SDS)-polyacrylamide gel.Expression of mMDP1 was confirmed by SDS-PAGE and western blot analysis as described previously 12 .www.nature.com/scientificreports/Diego, CA).Dead cells were stained using Fixable Aqua Dead Cell Strain kit (L34957, Thermo Fisher Scientific, Waltham, CA).Isolated PBMCs (1 × 10 6 ~ 2 × 10 6 /ml) were cultured with labeled mMDP1, G9.1 or a mixture of mMDP1 and G9.1 for 1 h at 5% CO2 and 37 °C.Cells were collected in tubes and treated with Human BD Fc Block (564219, BD Pharmingen, Franklin Lakes, NJ) to block Fc receptors, and then washed three timed with Staining medium (SM, Dulbecco's PBS containing 2% FBS and 0.05% NaN 3 ).Cell surface was stained with antibodies according to manufacturer's protocol, followed by fixation with 4% paraformaldehyde in SM overnight.

Statistics
Data was analyzed using Friedman tests followed by Dunn's multiple comparisons test.All analysis was performed using GraphPad Prism 9.5.1 (GraphPad Software, Boston, MA, USA) and results with p < 0.05 were considered significant and marked by asterisks.

Ethical approval
The experiments involving human blood collection were approved by the Niigata University Ethics Committee (approval number: 2015-2104) and were conducted in accordance with its guidelines.Informed consent was obtained from all participants.The experiments involving animals to obtain monoclonal antibodies were granted approval by the Niigata University Animal Experiment Committee (approved number: SA00073).The experiments were carried out in accordance with the committee's guidelines and followed the ARRIVE guidelines.

Figure 2 .
Figure2.The secondary structure and oligomerization of MDP1.(A) CD spectra of eMDP1 and mMDP1.eMDP1 and mMDP1 were adjusted to a concentration of 3.2 μM in 150 mM, 500 mM, and 1000 mM NaCl (pH 7.5).CD spectra of eMDP1 (left) and mMDP1 (right) are shown.The calculated percentage of α helix is indicated in the lower panel.(B) Sedimentation velocity measurement of eMDP1 and mMDP1.eMDP1 and mMDP1 were dialyzed against phosphate buffer (pH 7.0) containing 150 mM or 500 mM NaCl before the experiment.Radial fluorescence scans during sedimentation at 20 °C are depicted (dot colors indicate times in the following order: purple-blue-green-yellow-red).Solid lines represent the best-fit with a single species (left).The resulting sedimentation coefficient distributions are shown on the right.The peak of each graph was estimated to represent the monomer.Measurements were conducted with 150 mM NaCl (upper graphs) and 500 mM NaCl (lower graphs) for eMDP1, displayed on the four left columns, and for mMDP1 on the four right columns.(C) Glutaraldehyde crosslinking assay of eMDP1 and mMDP1.eMDP1 and mMDP1 in various molar concentrations of NaCl were incubated with or without glutaraldehyde (final concentration 0.2%) at room temperature for 30 min and then applied to SDS-PAGE.Four µM of eMDP1 and mMDP1 was run in the upper gel image, while 8 µM of each was run in the lower gel image.SDS-PAGE was performed by using a 4-15% gradient gel.Gel images on the left column show eMDP1, while those on the right show mMDP1.Monomer (black arrow), Dimer (red arrow), and Trimer (blue arrow) are indicated based on molecular weight.

Figure 3 .
Figure 3. IFN-gamma production induced by ex vivo stimulation of PBMC with eMDP1 or mMDP1.Peripheral blood mononuclear cells (PBMC) derived from three donors were plated at concentration of 2 × 10 5 cells per well and incubated with either 0.5 μM of eMDP1 or mMDP1, or untreated (none).The IFN-gamma production level of three days after culturing of two donors and five days after culturing of three donors is presented in the mean ± SD. *P < 0.05.Data were analyzed using Friedman tests followed by Dunn's multiple comparison test.Each bar represents the mean of triplicate values.Error bars, SD of the means.The results shown are representative of two experiments.

Figure 4 .
Figure 4. IFN-gamma productivity of mMDP1 and other M. tuberculosis proteins in BCG-vaccinated individuals.(A) Whole bloods samples obtained from five donors were incubated with 0.5 µM eMDP1, mMDP1 or, other M. tuberculosis antigens, including vaccine candidates such as MPT32, MPT45, MPT46, MPT53, MPT59, MPT63.After three days of cultivation, the concentration of IFN-gamma in culture supernatant was quantified using ELISA.Each bar represents the mean of triplicate values.Error bars, SD of the means.(B) The combined results of five donors are presented.Statistical significance: *, p < 0.05; mMDP1 versus none, eMDP1, MPT 46, and MPT 53, respectively.Data were analyzed using Friedman tests followed by Dunn's multiple comparison test.The results shown are representative of two experiments.

Figure 5 .
Figure 5. Post-translational modifications on mMDP1 lysine residues.Peptides generated by limited proteolysis of mMDP1 with trypsin were analyzed by mass spectrometry to determine the extent and localization of post-translational modifications.This included methylation, dimethylation, trimethylation, acetylation, and succinylation on all MDP1 lysine residues.The frequencies of each post-translational modification on lysin residues are depicted as heat maps.The values within each cell represent the ratio of post-translationally modified lysine to total lysine.

Figure 6 .
Figure 6.Evaluation of CpG ODNs as adjuvants for mMDP1.Whole blood from a donor was diluted with an equal volume of RPMI and supplemented with IL-2 (20 U/ml).The culture was conducted with 0.01, 0.05, 0.1, 0.5, and 1 μM of phosphodiester bonded ODNs either alone or in combination with mMDP1 (0.5 μM) for 3 days.The amount of released IFN-gamma was measured using ELISA.(A) IFN-gamma production when blood was cultured with ODNs alone (0.01 ~ 1 µM).(B) IFN-gamma production when whole blood was cultured with 0.5 μM ODNs only (blue columns) or in combination with mMDP1 (0.5 μM) (red columns).The blue line and green column represent IFN-gamma production when blood was cultured with 0.5 μM of mMDP1 alone.*, P < 0.05, indicating the comparison of IFN-gamma production stimulated by 0.5 µM of mMDP1 alone versus in combination of ODN + 0.5 µM mMDP1.Data were analyzed using Friedman tests followed by Dunn's multiple comparison test.(C) IFN-gamma production when blood was cultured with mMDP1 only (0.01 μM ~ 1 μM).Each bar represents the mean of triplicate values.Error bars, SD of the means.The results shown are representative of two experiments.

Figure 7 .
Figure 7. Augmented production of IFN-gamma and IFN-alpha when blood was stimulated with MDP1 and G9.1.Whole blood derived from six donors was cultured with MDP1 alone, or in combination with G9.1, or in combination with the negative form of G9.1(Neg G9.1).After three days of incubation with IL-2 at a concentration of 20 U/ml, the concentration of IFN-gamma (A) and IFN-alpha (B) in the culture supernatant were measured using ELISA.Statistical significance: *, p < 0.05.Data were analyzed using Friedman tests followed by Dunn's multiple comparison test.The results shown are representative of two experiments.

Figure 8 .
Figure 8. mMDP1 and G9.1 binding to immune cells populations.PBMCs (1 × 10 6 ~ 2 × 10 6 /ml) derived from a donor were cultured with Pacific Blue labeled mMDP1, Cy5 labeled G9.1 or a mixture of mMDP1 and G9.1 for 1 h at 5% CO2 and 37 °C.Next, the cell surface was stained with antibodies according to manufacturer's protocol and fixed with 4% paraformaldehyde in staining medium overnight.Cells were then analyzed the following day using the flow cytometer, NovoCyte 3000.(A) Analysis of the total cell population (left panel) and the monocyte-singlet region (right panel).(B) Cells that are positive for only mMDP1, G9.1, and both mMDP1 and G9.1 is highlighted with a red box (left column).These cells in the figure label "red box from the left column" were Lineage positive and HLA-DR positive antigen presenting cells (orange box in middle column), but negative for CD11c and CD303(pink box in right column).The green box indicates the dendritic cell region (right column).(C) Percentage of cells that are positive for mMDP1 or both mMDP1 and G9.1 in monocytesinglet region (red box in left panel of B).The results shown are representative of two experiments. https://doi.org/10.1038/s41598-024-58836-8